The localization of Oregon Green cadaverine-labeled thymosin beta(4), its fragments, and variants was investigated in cytoplasm-depleted A431 cells and in microinjected cells without and with fixation. The studied thymosin beta(4) variants included substitutions of the lysine residues within the basic cluster (14-KSKLKK-19) and the actin-binding motif (17-LKKTETQ-23). In contrast to Oregon Green cadaverine, none of the variants or fragments of thymosin beta(4) could pass the intact nuclear pore of cytoplasm-depleted cells and were hence excluded from the nucleus. However, an equal distribution of all thymosin beta(4) variants was observed in living cells. The nuclear localization is neither dependent on the actin-binding ability of thymosin beta(4) nor on its basic lysine cluster. The equal distribution of the beta-thymosins, the ability of the fragments thymosin beta(4)(1-26) and beta(4)(27-43) to enter the nucleus in intact cells immediately after injection, and their exclusion from cytoplasm-depleted nuclei make it unlikely that they are transported by a single transport protein. A passive but regulated diffusion could explain the described ability of thymosin beta(4) to shuttle into the nucleus.